Abstract: Adjuvant chemotherapy using 5-FU-based regimens reduces the risk of disease recurrence for patients with stage III and high-risk stage II colon cancer, however relapse occurs for 20-30% of patients. Thus, identifying factors that differentiate responsive from non-responsive disease and developing new treatments for non-responsive colorectal cancer (CRC) is essential. CRC is classified into four consensus molecular sub-types (CMS), and adjuvant chemotherapy with 5-FU-based regimens is the preferred treatment for most patients with CMS4 (stem-like) disease. However, even within this sub-type a substantial percentage of patients do not respond to 5-FU. We are developing polymeric fluoropyrimidines (FPs) that are more potent inhibitors of thymidylate synthase (TS), and that cause increased DNA damage through poisoning of DNA topoisomerase 1. Our first generation polymer, F10, displayed improved anti-cancer activity and reduced gastrointestinal tract toxicity relative to 5-FU in multiple pre-clinical models. Our 2nd generation polymer, capped F10 (CF10), displays improved plasma stability and greater tumor penetration than F10. CF10 is more than 400-fold more potent than 5-FU towards a sub-set of CRC cell lines in the NCI60 cell line panel. In a series of xenograft studies, CF10 displayed strong efficacy in CRC tumor models that are non-responsive to 5- FU. The goal of our project is to demonstrate CF10 is efficacious towards patient-derived CRC organoids that are non-responsive to 5-FU, thus providing a strong rationale for CF10 clinical evaluation. Our central hypothesis is that CF10, due to efficient conversion to DNA-directed metabolites, will be effective for treating CRC that is non-responsive to 5-FU-based regimens. In Aim 1, we assess CF10 efficacy in 5-FU- refractory CRC using: i) a panel of 20 CRC cell lines representative of all CRC subtypes; and ii) tumor organoids derived from 20 stage III CRC tumors from our NCI-designated tumor bank. Our studies will establish whether CF10 is effective for treating CRC sub-types that are non-responsive to 5-FU. In Aim 2, we develop a predictive classification model that stratifies response to CF10 based on sensitivity patterns in CRC cell lines and tumor organoids representing all CRC sub-types. CF10 GI50-related genes will be selected from the cell line data, and those genes that also correlate with response of tumor-derived organoids to CF10 based on RNASeq data will be used to construct an optimized classification model. We will validate the performance of the model on a 2nd, independent cohort of 12 stage III CRC samples sub-typed by RNASeq analysis and tested for CF10 responsiveness. We will also perform bioinformatics analyses to elucidate causal factors defining CF10 sensitivity. Our studies will develop a predictive classification model for CF10 sensitivity, and demonstrate its utility in predicting CF10 response to diverse CRC sub-types in patient-derived CRC organoids. Given the central role of FPs in treating CRC, successful development of CF10 for treating 5-FU non-responsive disease would be a major breakthrough with significant clinical implications.